Down syndrome, also referred to as Trisomy 21, is the most common congenital cause of severe mental retardation. Generally, fetal Down syndrome can be determined by diagnostic procedures including amniocentesis or chorionic villus sampling and karyotyping. However, these diagnostic procedures are invasive and involve risk to the woman and the fetus. For this and other reasons, amniocentesis or chorionic villus sampling and karyotyping are not routinely performed during all pregnancies. Instead, one or more screening methods may be utilized to determine when the risk to the pregnancy warrants the risk of undergoing an invasive diagnostic procedure.
The incidence of Down syndrome increases significantly with increasing maternal age. Historically, the prenatal detection of Down syndrome has focused on pregnant women at and over the age of 35, at which ages the risks of Down syndrome approach or exceed the risks of diagnostic procedures utilized to detect fetal Down syndrome. Therefore the standard method of prenatal screening has involved selecting women for diagnostic amniocentesis on the basis of maternal age. Age, however, is an inadequate screening criterion in that only about 20% of all Down syndrome pregnancies can be detected by carrying out amniocentesis and karyotyping on the 5% of pregnant women most at risk, that is, those aged 35 years or greater. And, because in actual clinical practice only about half of the women aged 35 years or greater undergo amniocentesis and karyotyping, fewer than 10% of Down syndrome pregnancies are prenatally detected.
In 1984 an association between lowered maternal blood alphafetoprotein (AFP) levels and fetal Down syndrome was discovered. For example, see "An association between low maternal serum alpha-fetoprotein and fetal chromosomal abnormalities"; Merkatz, Macri, et al.; Am. J. Obstet. Gynecol. 148:996, 1984; the disclosure of which is hereby incorporated by reference. In this publication it was noted that other chromosomal trisomies, in particular Trisomy 13 and Trisomy 18, were also associated with lowered maternal blood AFP levels. The incidence of these additional chromosomal trisomies (1 in 5000 pregnancies and 1 in 6600 pregnancies, respectively) is significantly lower than the general a priori risk associated with Trisomy 21 (Down syndrome, 1 in 800 pregnancies). However, because of the association of these other chromosomal trisomies with lowered MSAFP levels, and elevated or depressed free beta (HCG) levels, such abnormalities will also be detected within a screening protocol utilizing maternal blood AFP and free beta (HCG) and possibly additional markers described herein. It is obvious to those skilled in the art that in using the protocol described herein for Trisomy 21, the detection of Trisomy 13, Trisomy 18, Turners syndrome and other chromosomal anomalies may also be accomplished.
The association between lowered maternal blood AFP levels and fetal Down syndrome presented the opportunity to use a non-invasive blood screening test in the detection of Down syndrome cases in young, apparently unaffected families where approximately 80% of Down syndrome cases occur. It is estimated that the use of a screening test based on low maternal blood AFP (as a screening marker) would lead to the prenatal detection of approximately 20% of all cases of fetal Down syndrome.
Another method for screening-involves measuring the level of unconjugated estriol (UE) in maternal blood. For example, see "Maternal blood screening for Dwon syndrome in early pregnancy"; Wald, et al. British Journal of Obstetrics and Gynocology (BMJ) Volume 95, April 1988, the disclosure of which is hereby incorporated by reference.
More recently an association between elevated maternal blood levels of the Intact HCG molecule and the alpha subunit of HCG (HCG is composed of two subunits) and fetal Down syndrome was discovered. For example, see "Abnormal Maternal Serum Chorionic Gonadotropin Levels in Pregnancies with Fetal Chromosome Abnormalities"; Bogart, Pandian and Jones; Prenatal Diagnosis, Vol. 7, 623-630 (1987) the disclosure of which is hereby incorporated by reference. In the Bogart article it is estimated that the use of elevated maternal blood Intact HCG levels and elevated maternal blood levels of the alpha subunit of HCG, would detect approximately 68% of the chromosomally abnormal fetuses. However, these results were obtained from a study on pregnancies at 18-25 weeks of gestation and the affected cases appear to be of women previously identified as being at risk for Down syndrome.
U.S. Pat. No. 4,874,693 to Bogart discloses an association between elevated maternal blood HCG levels and elevated maternal blood levels of the alpha subunit of HCG, in weeks 18-25 of pregnancy and fetal Down syndrome. In the Bogart patent it is estimated that the use of elevated maternal blood HCG levels and elevated maternal blood levels of the alpha subunit of HCG in a screening protocol, would detect a greater percentage of chromosomally abnormal fetuses than the use of AFP or UE alone. In a paper entitled "Human Chorionic Gonadotropin Levels in Pregnancies with Aneuploid Fetuses" (Bogart et al., Prenatal Diagnosis, Vol. 9, 379-384 (1989)), Bogart discloses that a screening method utilizing HCG and the alpha subunit of HCG is not useful at 9-11 weeks gestation (the first trimester of pregnancy) for selecting pregnancies at risk for fetal aneuploidy (including Down syndrome).
Generally, as suggested above, screening by evaluation of maternal blood HCG has involved only the measurement of the Intact HCG molecule and additionally the measurement of the alpha subunit of HCG. Although these screening methods do detect fetal Down syndrome, there is a need and a desire for a method which detects a greater percentage of fetal Down syndrome cases.
I have discovered a previously unknown association between elevated levels of maternal blood free beta (HCG) and fetal Down syndrome. I have also discovered a previously unknown association between the maternal blood level of free beta (HCG) and the maternal blood level of AFP and fetal Down syndrome. I have further discovered a previously unknown association between the ratio of the maternal blood level of free beta (HCG) to the maternal blood level of the intact HCG molecule and fetal Down syndrome. I have still further discovered that using a multivariate discriminant analysis technique improves the detection efficiency of a screening method using the maternal blood level of free beta (HCG), or the maternal blood level of free beta (HCG) and the maternal blood level of AFP, or the log of either, or the log of both, especially when gestational age is also incorporated as a variable in the discriminant analysis technique, for a chosen risk cut-off level. Gestational age refers to the age of the pregnant woman's fetus. Detection efficiency refers to the percentage of cases of fetal Down syndrome which are correctly detected for a chosen risk cut off level. The risk cut off level will be more fully explained in a following section. Discriminant analysis is a generally known approach to multivariate analysis involving the separation of a population into two or more groups on the basis of probability. Discriminant analysis is also sometimes described as a way of constructing a linear combination of independent variables, thus reducing the problem of measuring group differences to a univariate problem. A general discussion of discriminant analysis can be found in Marketing Research; Churchill, G.A.; Dryden, 1976; Chapter 15, pages 530-543, the disclosure of which is hereby incorporated by reference. I have discovered that subjecting the maternal blood levels of free beta (HCG), the maternal blood levels of intact HCG, the ratio of the maternal blood level of free beta (HCG) to the maternal blood level of the intact HCG molecule, the maternal blood level of AFP, the maternal blood level of UE, and gestational age to multi-variate discriminant analysis detects a greater percentage, with a lower false positive rate, of fetal Down syndrome cases than any other known screening method for the prenatal detection of Down syndrome. I have further discovered that a still greater number the cases of fetal Down syndrome may be detected by using only the measurements of the maternal blood levels of free beta (HCG) and the maternal blood levels of AFP and subjecting the log of each measurement and gestational age to a multivariate discriminant analysis. These and other discoveries will be more fully explained in the Summary of the Invention section and the Detailed Description of the Invention section.
One object of the present invention is to provide a method and process for screening for fetal Down syndrome which detects a greater percentage of fetal Down syndrome cases with a given false positive rate than other known prenatal screening methods.
Another object of the present invention is to provide a method and process for screening for fetal Down syndrome which has a lesser false positive rate for a given detection percentage than other known methods.
A still further object of the present invention is to apply multi-variate discriminant analysis to methods for screening for Down syndrome to detect a greater percentage of fetal Down syndrome cases with a lesser false positive rate.
A further object of the present invention is to provide a method and process for screening for fetal Down syndrome by measuring the level of maternal blood free beta (HCG).
A still further object of the present invention is to provide a method and process for screening for fetal Down syndrome by measuring the maternal blood level of AFP and the maternal blood level of free beta (HCG).
Other objects and advantages of the present invention will become apparent in the following description of the invention.